Food Research International 51 (2013) 7585

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

Xylo-oligosaccharides from lignocellulosic materials: Chemical structure, health

benets and production by chemical and enzymatic hydrolysis
Ana Flvia Azevedo Carvalho a,b,, Pedro de Oliva Neto b, Douglas Fernandes da Silva b, Glucia Maria Pastore a
a
b

Department of Food Science, School of Food Engineering, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, ZIP code 13083-862, Campinas, SP, Brazil
Department of Biological Sciences, School of Science and Letters, So Paulo State University (UNESP), Rua Dom Antonio, 2100, ZIP code 19806-380, Assis, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 27 June 2012
Accepted 20 November 2012
Keywords:
Lignocellulosic materials
Chemical and enzymatic hydrolysis
Xylanases
Xylo-oligosaccharides

a b s t r a c t
Currently, there is worldwide interest in the technological use of agro-industrial residues as a renewable
source of food and biofuels. Lignocellulosic materials (LCMs) are a rich source of cellulose and hemicellulose.
Hemicellulose is rich in xylan, a polysaccharide used to develop technology for producing alcohol, xylose, xylitol and xylo-oligosaccharides (XOSs). The XOSs are unusual oligosaccharides whose main constituent is xylose linked by 14 bonds. The XOS applications described in this paper highlight that they are considered
soluble dietary bers that have prebiotic activity, favoring the improvement of bowel functions and immune
function and having antimicrobial and other health benets. These effects open a new perspective on potential applications for animal production and human consumption. The raw materials that are rich in hemicellulose include sugar cane bagasse, corncobs, rice husks, olive pits, barley straw, tobacco stalk, cotton stalk,
sunower stalk and wheat straw. The XOS-yielding treatments that have been studied include acid hydrolysis, alkaline hydrolysis, auto-hydrolysis and enzymatic hydrolysis, but the breaking of bonds present in these
compounds is relatively difcult and costly, thus limiting the production of XOS. To obviate this limitation, a
thorough evaluation of the most convenient methods and the opportunities for innovation in this area is
needed. Another challenge is the screening and taxonomy of microorganisms that produce the xylanolytic
complex and enzymes and reaction mechanisms involved. Among the standing out microorganisms involved
in lignocellulose degradation are Trichoderma harzianum, Cellulosimicrobium cellulans, Penicillium janczewskii,
Penicillium echinulatu, Trichoderma reesei and Aspergillus awamori. The enzyme complex predominantly
comprises endoxylanase and enzymes that remove hemicellulose side groups such as the acetyl group. The
complex has low -xylosidase activities because -xylosidase stimulates the production of xylose instead
of XOS; xylose, in turn, inhibits the enzymes that produce XOS. The enzymatic conversion of xylan in XOS
is the preferred route for the food industries because of problems associated with chemical technologies
(e.g., acid hydrolysis) due to the release of toxic and undesired products, such as furfural. The improvement
of the bioprocess for XOS production and its benets for several applications are discussed in this study.
2012 Elsevier Ltd. All rights reserved.

Contents
1.
2.
3.
4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . .
Chemical structure of xylo-oligosaccharides . . . . . . . .
Health benets of xylo-oligosaccharides . . . . . . . . .
Soluble bers as substitutes for antibiotics in feed . . . . .
Technologies for obtaining xylo-oligosaccharides . . . . .
5.1.
Pretreatments for xylan extraction . . . . . . . . .
5.1.1.
Autohydrolysis . . . . . . . . . . . . . .
5.1.2.
Alkaline and acid pretreatments . . . . . .
5.2.
Enzymatic treatment of pre-hydrolyzed LCM residues
5.2.1.
Xylanases . . . . . . . . . . . . . . . .
5.3.
Enzymatic technology for XOS production . . . . .

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XOS production
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Corresponding author at: So Paulo State University (UNESP), Rua Dom Antonio, 2100, ZIP code 19806-380, Assis, SP, Brazil. Tel./fax: +55 1833025848x5716.
E-mail addresses: anafbio@yahoo.com.br (A.F.A. Carvalho), poliva@assis.unesp.br (P.O. Neto), douglasfsilva@gmail.com (D.F. da Silva), glaupast@fea.unicamp.br (G.M. Pastore).
0963-9969/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2012.11.021

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A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

6.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

1. Introduction
Currently, one of the greatest challenges is to increase the production of healthy and economically affordable foods that are available
for a large proportion of the world's population. The increase in
food production must be sustainable and avoid the deforestation of
new areas that are currently occupied by forests. In this regard, research into the use of agro-industrial residues for food production
should be intensied because these wastes are underutilized and billions of tons are discarded annually.
Lignocellulosic materials (LCM) represent the most abundant organic residues in the world. Considering only the sugar and ethanol
in Brazil, the estimated production of sugar cane for the 2012/13
season will be 596.6 billion tons (CONAB, 2012). At 135 kg of dried
bagasse per ton of crushed cane (Brienzo, Siqueira, & Milagres,
2009), the total bagasse generated in this season will be approximately 80.5 billion tons.
Cane bagasse is currently used in sugar mills for burning in boilers,
generating steam, heating cane juice and operating the mills, thus
saving energy in the process. Some sugar mills also use cane bagasse
to co-generate electricity. However, some cane bagasse remains that
can be used for other purposes, especially to produce products with
high added value.
Sugar cane bagasse has 2429% hemicellulose, represented by
1-arabino-(4-0-methyl-D-glucurono)-D-xylan, as well as cellulose,
lignin, minerals, waxes and other compounds (Brienzo et al., 2009;
Gottschalk, Oliveira, & Bom, 2010; Pandey, Soccol, Nigam, & Soccol,
2000; Rodrigues et al., 2010; Song & Wei, 2010). Hemicellulose is a
heteropolysaccharide with xylan as the major carbohydrate and
mainly comprises polymers of xylose, arabinose, galactose, mannose
and glucose (Garrote, Domnguez, & Paraj, 1999).
Xylan is also a heteropolysaccharide with a backbone formed by
xylose homopolymer subunits (Nabarlatz, Ebringerov, & Montan,
2007; Rodrigues et al., 2010; Saha, 2003; Santos, Sarrouh, Rivaldi,
Converti, & Silva, 2008).
In other LCM residues such as tobacco stalk, cotton stalk, sunower stalk and wheat stalk, the main chemical composition is 1921%
xylan, 3344% cellulose and 2029% lignins (Akpinar, Erdogan, &
Bostanci, 2009a,b). Reports on the chemical composition of corncobs,
one of the most important LCMs studied for xylo-oligosaccharides
(XOS) production, suggest the presence of glucan 3135%, lignin
1014% and a high xylan content (3035%) (Garrote, Dominguez, &
Paraj, 2002; Tan et al., 2008; Yang, Xu, Wang, & Yang, 2005).
Samanta, Senani, et al. (2012) found similar results for corncobs:
27.7% cellulose, 38.8% xylan, 9.4% lignin, 75.9% neutral detergent
ber and 37.1% acid detergent ber.
In recent years, there has been increasing interest in new prebiotics such as XOS. XOSs are produced by hydrolyzing xylan. Commercialized as a white powder, XOSs are oligomers containing two
to ten xylose molecules linked by 14 bonds (Mkelinen et al.,
2010). XOSs are considered non-digestible oligosaccharides (NDOs),
non-cariogenic in humans and have important biological properties.
They are used as dietary sweeteners in low-calorie diet foods and for consumption by individuals with diabetes (Choque Delgado, Tamashiro,
Junior, Moreno, & Pastore, 2011; Rivero-Urgell & Santamaria-Orleans,
2001; Vzquez et al., 2000).
Some prebiotics such as fructo-oligosaccharides and mannan oligosaccharides are currently widely used in feed (for pigs, chickens, cattle
and pets), replacing the use of antibiotic growth promoters (AGPs).
Due to the overuse of antibiotics, virulent strains of pathogens are

increasing, and there is the possibility of antibiotic residues in meat.

Consequently, beginning in 2006, the European Union banned the use
of growth promoting antibiotics (GPAs) in farming (Falco et al.,
2007; Wallace, 2007). Therefore, the search for new molecules with
powerful prebiotic activity is essential, including the emergent prebiotic
XOS (Olano-Martin, Gibson, & Rastall, 2002; Rastall & Maitin, 2002;
Rycroft, Jones, Gibson, & Rastall, 2001).
XOS is a promising oligosaccharide class that stimulates increased
levels of bidobacteria to a greater extent than does FOS (Tuohy,
Rouzaud, Brck, & Gibson, 2005) or other oligosaccharides (Rycroft
et al., 2001; Samanta, Senani, et al., 2012).
XOS production from LCM is not simple or economical because
it depends on two treatment steps. The rst step is the xylan extraction from LCM, which includes a chemical pretreatment. Although
there are multiple treatments for xylan extraction, there is no consensus favorite among them. The pretreatment methods discussed
in this paper are autohydrolysis and acid and alkaline hydrolysis.
The second step includes the xylanase enzymatic reaction or the
acid hydrolysis of xylan. Therefore, developing an efcient and economical xylanase production bioprocess for use in XOS production
is necessary.
The biochemical characterization of XOS-producing microorganisms, their modes of action and their xylanase activities are included
in the present review. The oligosaccharide production by enzymatic
processes is inuenced by some critical factors. The yield, productivity and type of oligosaccharide produced depend on the type of enzyme, its microorganism source and the stability and activity of the
xylanase at different pHs and temperatures.
Furthermore, the free enzyme can be produced and used in mixtures with the reaction substrate, immobilized and recycled, or the
enzyme can be produced in situ via fermentation. The type and concentration of substrate, the bioprocess parameters (such as reaction
time, temperature and pH), the type and conditions of pretreatment
for xylan extraction and downstream processes are considered in
this study, aiming to contribute to improvements in XOS production
technology.

2. Chemical structure of xylo-oligosaccharides

XOSs are oligosaccharides commercialized as a white powder
containing two to ten xylose molecules linked by 14 bonds
(Fig. 1), but molecules with degree of polymerization (DP) 20
have been considered XOS (Mkelinen et al., 2010; Moure, Gulln,
Domnguez, & Paraj, 2006; Vzquez et al., 2000).
XOS stabilities can differ greatly depending on the types of oligosaccharide and sugar residues, linkages, ring forms and anomeric congurations. Generally, -linkages are stronger than -linkages, and
hexoses are more strongly linked than pentoses. Most oligosaccharides can be hydrolyzed, resulting in the loss of nutritional and physicochemical properties at pH b 4.0, when treated at high temperatures
for short time periods, or when subjected to prolonged storage under
room conditions. For example, fructooligosaccharides (FOS) it is related that in a 10% solution of pH 3.5 less than 10% is hydrolyzed after
heat treatments of 5 min at 45 C or 60 min at 70 C. After two days
at 30 C less than 50% is hydrolyzed (Voragen, 1998).
Nevertheless, the XOSs are stable over a wide range of pHs (2.5
8.0), even the relatively low pH value of gastric juice, and temperatures up to 100 C. This is an advantage compared with other NDOs
such as FOS and inulin (Bhat, 1998).

A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

HO

X1 = xylose

OH

HO

77

OH

HO
HO

OH

X2 = xylobiose

HO

OH
O

OH

HO
HO

OH

O
O

HO

OH

HO

OH

OH

n = 1:
n = 2:
n = 3:
n = 4:
n = 5:

X3 = xylotriose
X4 = xylotetraose
X5 = xylopentaose
X6 = xylohexaose
X7 = xyloheptaose

Fig. 1. Schematic structure of xylose and xylo-oligosaccharides.

3. Health benets of xylo-oligosaccharides

XOS are not digested by humans because the human body lacks
the enzymes required to hydrolyze the -links, so they are considered
prebiotics and soluble ber because they are not degraded in the
stomach and reach the large intestine intact. Therefore, they are
classied as NDOs, and they show several health benets (Table 1).
This property allows XOSs to be used as dietary sweeteners for
low-calorie diet foods and for consumption by individuals with diabetes (Choque Delgado et al., 2011; Nabarlatz et al., 2007; Rivero-Urgell
& Santamaria-Orleans, 2001; Vzquez et al., 2000). NDOs are noncariogenic because they are not utilized by mouth microbiota
(Vzquez et al., 2000).
The biological activity of a XOS depends on its molecular weight
distribution (Hughes et al., 2007). XOSs with fewer than four monomer units are important for prebiotic applications because they promote the proliferation of bidobacteria, which are considered as
benecial microorganisms in the human intestine (Gulln et al.,
2008).
XOSs provide health benets as the active ingredients in functional foods. These components may be present in food or as added industrial products (Nabarlatz et al., 2007; Vzquez et al., 2000).
The addition of XOS to food adds physiological properties benecial for the body, including improvement in bowel function, calcium
absorption and lipid metabolism, prevention of dental caries, protection against cardiovascular disease and reduction in the risk of colon
cancer due to the formation of short-chain fatty acids (Grootaert
et al., 2007; Wang et al., 2009). In addition, XOS provides benecial
effects on: skin, blood, immunological system, anti-oxidant, antiinammatory effects and antiallergic activities (Aachary & Prapulla,
2009). XOS can improve the microbiota increasing the presence of
benecial bacteria, particularly Bidobacterium, that inhibit the
growth of pathogenic and putrefactive bacteria (Mussatto & Mancilha,

2007). Some studies indicate that 4 g of XOS per day for 3 weeks improved the intestinal microbiota among people who are above
65 years old (Chung et al., 2007).
XOS also have favorable technological features, including stability
at acidic pH, heat resistance, the ability to achieve signicant biological effects at low daily doses, low calorie content, non-toxicity and
other properties that have yet to be studied (Vzquez et al., 2000).
4. Soluble bers as substitutes for antibiotics in feed
The use of antibiotic growth promoters (AGPs) in animal production began half a century ago when residues of chlortetracycline production were added to chicken feed. These residues were added to
serve as a source of vitamin B12, but they caused a growth stimulation that was far too large to be explained as only a vitamin effect
(Brezoen, Van Haren, & Hanekamp, 1999). This observation was
quickly extended to other antibiotics and other animal species, leading to widespread adoption of AGP inclusion in feeds. In recent decades, considerable amounts of antibiotics have been used in animal
production, as both therapeutics and growth promoting agents
(Falco et al., 2007).
Antibiotics have been widely used in animal production, not only
to treat or prevent infection in farm animals but also for low
dose-long term administration, usually given in feed, to help animals
gain weight more quickly and increase the productivity and yield in
animal production. However, overuse of antibiotics encourages resistance and the production of more virulent strains of pathogens. Consequently, beginning in 2006, the European Union banned the use of
growth promoting antibiotics (GPAs) in farming (Falco et al., 2007;
Wallace, 2007).
Mannan oligosaccharides (MOS) act mainly by preventing colonization more than by stimulating benecial microorganisms, and they are
considered important prebiotics. Many pathogens have mbriae that

Table 1
Xylo-oligosaccharide benets for the healthy human.
XOS Benets

References

Modulation of the colon microbiota increasing the number of bidobacteria and Lactobacillus.

Gulln et al. (2008), Moure et al. (2006), Van Laere, Hartemink,

Bosveld, Schols, and Voragen (2000)
Mussatto and Mancilha (2007)
Blaut (2002), Grootaert et al. (2007), Wang et al. (2009)

Decrease in the number of pathogenic and putrefactive bacteria.

Growth of health-promoting bacteria in the intestinal tract leading to the production of short chain
fatty acids, which cause benecial effects for metabolism (e.g., reduction of osteoporosis). Protection
against cardiovascular disease and reduction of the risk of colon cancer due to the formation of
these acids.
Improvements in bowel function and calcium absorption; the prevention of dental caries.
Biological effects at low doses. Effects related to skin and blood, immunological action, anti-oxidant
activity, anti-inammatory and antiallergic action.
Low calorie.

Grootaert et al. (2007), Wang et al. (2009)

Aachary and Prapulla (2009), Vzquez et al. (2000)
Vzquez et al. (2000)

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A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

specically attach to the mannose residues of intestinal cell receptors,

but otherwise, if they bind to mannose residues of MOS they will not
attach to the mucosa. In several animals trials, MOS performances
were comparable to those obtained with AGPs (Fonseca, Falco,
Kocher, & Spring, 2004; Mouro et al., 2006; Pinheiro et al., 2004).
Mouro et al. (2006), working with rabbits, reported that MOS increased the length of ileal villi, possibly as a result of the reduction in
microbial counts, which they also detected. Several studies have
shown a protective effect of prebiotics in animals infected with typical
food borne bacteria, including Salmonella, Escherichia coli and Listeria
monocytogenes. (Gill, Shu, Lin, Rutherfurd, & Cross, 2001; LeBlanc,
Castillo, & Perdigon, 2010; Shu et al., 2000; Silva et al., 2004; Truusalu
et al., 2008; Vieira et al., 2008). According to Licht, Ebersbach, and
Frkir (2012), anti-infective activities of prebiotics have been studied
in mice, rats, pigs, broilers, hamsters and guinea pigs and generally
showed that prebiotics improved host resistance to bacterial infections,
contributed to diarrhea prevention, decreased microbial colonization
and translocation of the pathogen, and increased animal survival.
However, according to Falco et al. (2007), there is a lack of consistency in the results obtained with prebiotics. This inconsistency
can be explained by differences in experimental protocols, e.g., the
number of animals, hygienic conditions and the nature and amount
of the prebiotic added to feed. In addition, it is possible that prebiotics
show benets in long-living beings, but do not in short-lived species,
such as rabbits. Mouro et al. (2006) conrmed that the amount of
prebiotics required for efcient animal production could limit its
use due to high cost.
Fructooligosaccharides, inulin, lactulose and galactooligosaccharides
are well established as prebiotics in Europe, Japan and the United States.
However, a much wider range of emergent prebiotics exists, which includes soyoligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, gentiooligosaccharides, lactosucrose, glucooligosaccharides and
pecticoligosaccharides (Olano-Martin et al., 2002; Rastall & Maitin,
2002; Rycroft et al., 2001). Much of the supportive evidence for these
new prebiotics has been generated by researchers in Japan and, increasingly, by groups in Europe (Tuohy et al., 2005).
XOS can be effectively fermented by bidobacteria (Jaskari et
al., 1998; Moura et al., 2008; Okazaki, Fujikawa, & Matsumoto,
1990). Rycroft et al. (2001) showed that XOS as the sole carbon
source resulted in a signicant increase in bidobacterial numbers
after 24 h (Sharp, Fishbain, & Macfarlane, 2001). The bidogenic
nature of XOS has been conrmed in rats. Campbell, Fahey, and
Wolf (1997) fed rats a 6% (w/w) diet of XOS and reported a significant increase in both cecal and fecal bidobacteria. The XOS diet
stimulated the increase in bidobacteria levels to a greater extent
than did FOS (Tuohy et al., 2005) and other oligosaccharides
(Rycroft et al., 2001).
Samanta, Senani, et al. (2012) studied the in vitro effect of crude
XOS (from corncob xylan) on the growth of the following probiotics
responsible for improvement of digestibility in ruminants gut: Enterococcus fecalis CCD10, Enterococcus faecium TCD3, Lactobacilllus
maltromicus MTCC108 and Lactobacillus viridiscens NCIM2167. These
bacteria satised the major probiotic criteria (Senani et al., 2009).
The organisms were grown in MRS broth supplemented with 1%
(w/v) glucose or 1% FOS (Sigma, USA) or 1% crude XOS produced by
the authors. Crude XOSs were capable to growth probiotic organisms
as compared to either control or 1% glucose. The bacterial growth was
higher with E. faecium, followed by E. fecalis, L. maltromicus, and
L. viridiscens. The enhanced growth of E. faecium indicated that this
bacteria possessed enzymes for hydrolyzing the glycosidic bonds
present in the XOS. However, the inuence of FOS on the growth
stimulation of probiotics was higher than either XOS or glucose. The
authors believe these quantitative differences may be due to the
crude preparation of XOS compared against pure FOS.
According to review of Otieno and Ahring (2012a) XOSs have been
recently regarded as emerging prebiotics that may present the same

or more desirable properties than the more established oligosaccharides. Other studies have been showed the XOS ability in the growth
of some benecial bacteria. The growth of Lactobacillus brevis and
their capability to utilize XOS from corncob as carbon and energy
source was proved (Moura et al., 2007).
XOSs were obtained by autohydrolysis of rice husk and puried
by nanoltration and ion exchange processing. They were evaluated
in a medium for promoting the growth of Bidobacterium adolescentis
CECT 5781, Bidobacterium longum CECT 4503, Bidobacterium
infantis CECT 4551 and Bidobacterium breve CECT 4839. All of them
grew on XOS medium, but the specic growth rate of B. adolescentis
(0.58 h 1) was higher than the ones determined for B. longum,
B. infantis and B breve (0.37, 0.30 and 0.40 h 1, respectively). The percentage of total XOS by B. adolescentis was 77% in 24 h of culture and
the highest percentage of consume corresponded to xylotriose
(90%), followed by xylobiose (84%), xylotetraose (83%) and
xylopentaose (71%) (Gulln et al., 2008). On the other hand, XOS
shows an ability for suppressing the growth of Clostridium and bacteriostatic action against Vibrio anguillarum (Izumi & Azumi, 2001).
5. Technologies for obtaining xylo-oligosaccharides
5.1. Pretreatments for xylan extraction
Various pretreatments have been used for the complete extraction
of hemicellulose to make xylan for enzymatic reactions. The pretreatments include thermal and chemical methods (Aachary & Prapulla,
2009).
Before starting the pretreatment, the raw material can be prepared by washing with ethanol or ethyl acetate to remove impurities
and other compounds (e.g., waxes and pectin) and make the later
XOS-producing steps easier (Brienzo et al., 2009; Vzquez et al.,
2005). Furthermore, alcohols and ketones can be used in the recovery
of soluble xylan or to concentrate the released XOS (Akpinar et al.,
2009b; Vzquez et al., 2005).
The current methods used for pretreatments are autohydrolysis,
acid or alkaline pre-hydrolysis and enzymatic hydrolysis.
5.1.1. Autohydrolysis
Autohydrolysis involves the deacetylation of xylan by the thermal
hydrolysis of hemicellulose to produce acetic acid (Garrote et al.,
1999, 2002; Kabel et al., 2002). The products obtained from LCM by
this process contain a variety of undesirable components such as lignin, monosaccharides, furfural, and others, thereby requiring further
purication (Zhu, Wu, Zhang, Li, & Gao, 2006).
XOS produced by autohydrolysis contains a signicant proportion
of compounds with a high degree of polymerization (Nabarlatz et al.,
2007). This treatment can be used as a rst step in the fractionation
processes for LCM.
After prehydrolysis, the LCM can be separated into two fractions.
The soluble and liquid phase is rich in hemicellulose, and it can be
decomposed into valuable products, such as soluble oligosaccharides,
sugars and aldehydes. The precipitated solids are high in cellulose and
lignin, and they can be separated for further processing to enable a
variety of possible applications, including the production of cellulose
pulp (Caparrs, Ariza, Garrote, Lpez, & Daz, 2007) and fermentable
sugars or fuel (Yez et al., 2006).
When LCM containing xylan is submitted to autohydrolysis under
mild operating conditions, xylo-oligosaccharides (XOSs) are the main
products of the hydrolysis of hemicellulose. To obtain XOS with a degree of polymerization of 26 xylose units, the products released by
autohydrolysis require extra treatment, e.g., enzymatic or acid hydrolysis (Moure et al., 2006). Moreover, autohydrolysis requires specialized equipment that needs to be operated at high temperatures (Zhu
et al., 2006).

A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

5.1.2. Alkaline and acid pretreatments

The exposure of hemicellulose to enzymatic hydrolysis must frequently be preceded by pretreatment with acid or alkali (Fig. 2).
However, these extractions must be mild to limit the amount of xylose released by the acid treatment and avoid or decrease the
resulting corrosion and pollution. Experiments carried out with corncob demonstrated that acid pretreatment alone did not alter the
xylanlignin complex but the subsequent baking did modify this
structure, allowing the removal of lignin and production of XOS
after enzymatic hydrolysis (Yang et al., 2005).
The evaluation of pretreatment methods is based on two criteria:
xylan content after pretreatment and quantities of XOS produced
under certain standard conditions.
Acid and alkaline pretreatments of corncob were compared, with
the acid pretreatment followed by cooking. The alkaline prehydrolysis showed the best performance in exposing xylan to the action of endoxylanases, resulting in a high yield of XOS (Akpinar,
Erdogan, Bakir, & Yilmaz, 2010). The alkaline pre-hydrolysis has the
disadvantage of not preserving the acetyl groups present in xylan
(Nabarlatz et al., 2007; Vzquez et al., 2005), which may limited the
water solubility of XOS (Nabarlatz et al., 2007). However, alkaline
pretreatment with hydrogen peroxide in sugar cane bagasse showed
good efciency in delignication and hemicellulose solubilization,
preserving its chemical structure (Brienzo et al., 2009; Sun, Sun,
Sun, & Su, 2004).
Different methods of corncob pretreatment were evaluated to
make the xylan more accessible to the xylanase reaction and for the
complete extraction of the xylan. The xylan extraction yield from
raw corncob (without any pretreatment) is about was 31.9%. When
the alkaline, acid and pressure pretreatments were used, these yields
increased to 40.8, 39.2 and 40.0%, respectively (Aachary & Prapulla,
2009). These results justify the use of pretreatment before xylanase
reaction of crude xylan, since there was an increase of 18.6% in
xylan extraction yield.
When the alkaline, acid (0.01 M H2SO4 at 60 C in 12 h) and pressure steam cooked (135 C/30 min) pretreatments were compared
using the reaction of xylan extracted by a commercial xylanase
(Table 2), the XOS yields and concentrations, were 89% (2.06 mg/
ml), 81% (5.8 mg/ml) and 77% (3.7 mg/ml), respectively. Therefore,
if the xylan extraction yields were similar (approximately 40%), the
best pretreatment according to the XOS yield was alkaline (Aachary
& Prapulla, 2009). These last pretreatments were superior to an acid
process since the XOS yield was considerably lower (52%) (Aachary
& Prapulla, 2009). Although the acid concentration used was low,

79

the reaction time was long, and probably allowed the degradation
of xylan to xylose under these conditions.
The effect of different acid concentrations (1.22%4.90% H2SO4) on
XOS production from xylans of tobacco stalk (TSX), cotton stalk
(CSX), sunower stalk (SSX) and wheat stalk (WSX) was evaluated
(Akpinar et al., 2009b). Reducing sugar production rates increased
with time and acid concentration.4.9% sulfuric acid at 100 C produced mainly monosaccharides rather than oligosaccharides. The
best conditions for acid hydrolysis were 2.45% H2SO4 in 30 min of reaction time at 100 C. However even under these condition the XOS
yield was low for tobacco (13%), cotton stalk (7.5%), sunower stalk
(12.6%) and wheat straw (10.2%).
More recently, Otieno and Ahring (2012b) obtained high yield of
XOS from switchgrass (Panicum virgatum 84.1%), morning light
(Miscanthus sinensis 64.9%) and cane bagasse (92.2%) using rapid
thermo-acid process (0.1% H2SO4 at 145 C for 1 h). The content
(19.7%) and quality of XOS (70% X2 to X4) from cane bagasse was
also interesting and xylose content was not high (5.9%) when it was
compared with enzymatic hydrolysis (22.5%) (Brienzo et al., 2010).
The rapid thermo-acid treatment of sugarcane bagasse seems to be
also more interesting for the XOS yield (92%) when compared to
the yield of 3940% by enzymatic method (Brienzo et al., 2010;
Carvalho, Oliva-Neto, & Pastore, 2012a,b). Furthermore, the rst
treatment is simpler in the hydrolysis step. The major impediment
of the acid treatment is the production of toxic components (furfural
and HMF) that require a greater degree of purication.
The type and chemical structure of the substrate and the enzyme
specicity are important factors in the enzymatic hydrolysis of
xylan to produce XOS.
The XOS yield from enzymatic hydrolysis using xylan extracted
from tobacco stalk by alkaline pretreatment (11.4%) was slightly
lower than from acid hydrolysis. However, two of the major disadvantages of acid hydrolysis are the production of considerably high
amounts of monosaccharides and the formation of undesirable toxic
furfural (Akpinar et al., 2010). These last products make purication
steps more difcult and expensive in addition to decrease in the
XOS yield. If the importance of pretreatment and the limitation of
acid hydrolysis is to be proven, the best procedure is to use alkaline
and thermal hydrolysis. An extraction from corncob using 2% and
12% alkali and steam hydrolysis improved the xylan recovery
from12.5% to 63.2% (for potassium hydroxide) and 14.4% to 83.5%
(for sodium hydroxide), respectively. These results showed the importance of higher alkali concentrations in hydrolysis, with increased
xylan recovery at increased alkali levels. Xylan obtained by this

Fig. 2. Some alternative chemical pretreatments and acid or enzymatic treatments for XOS production from lignocellulosic residues.

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A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

Table 2
Sources of LCM residues, pretreatments for xylan extraction, and different treatments for XOS production.
LCM residue

Pretreatment
xylan
extraction

Corncob
Alkali pretreat.
Corncob
Acid pretreat.
Corncob
Steam cooked
Palm oil
Autohydrolysis
Tobacco stalk
Alkali pretreat.
Sunower stalk
Alkali pretreat.
Cotton stalk
Alkali pretreat.
Wheat straw
Alkali pretreat.
Tobacco stalk
Alkali pretreat.
Corncob
Alkali pretreat.
Cane bagasse
Alkali pretreat.
Corncob
Alkali pretreat.
Corncob
Alkali pretreat.
Corncob
Alkali pretreat.
Triploid Populus tomentosa Alkali pretreat.
Cane bagasse
Alkali pretreat.

Reference
Reaction time XOS production XOS
Treatment XOS production Xylan
yield (%)
production
extraction for XOS
(g/l)
production
yield (%)
Enzymatic hydrolysis

Enzymatic hydrolysis
Enzymatic hydrolysis
Enzymatic hydrolysis
enzymatic hydrolysis
Enzymatic hydrolysis
Enzymatic hydrolysis
Acid hydrolysis
Enzymatic hydrolysis
Enzymatic hydrolysis
Acid hydrolysis
Acid hydrolysis
Acid hydrolysis
Enzymatic hydrolysis
Enzymatic hydrolysis

40.8
39.2
40.0

21.8
18.9
21.3
20.6
20.0
17.9
86.0
83.5
83.5
83.5

85.0

24 h
24 h
24 h
24 h
24 h
24 h
24 h
24 h
30 min
8h
96 h
15 min
30 min
60 min
14 h
8h

method was submitted to 0.25 M sulfuric acid hydrolysis at 90 C

for XOS production. XOS yields of 86.6% (0.65 mg/ml), 82.4%
(0.79 mg/ml) and 68.5% (0.88 mg/ml) in 15, 30 and 60 min (Table 2)
were obtained, respectively (Samanta, Jayapal, et al., 2012). According
to these results, it seems more convenient to use weak acids only during
the xylan hydrolysis for XOS production and not in the xylan extraction
step.
5.2. Enzymatic treatment of pre-hydrolyzed LCM residues and XOS
production
5.2.1. Xylanases
5.2.1.1. Microorganisms that produce of xylanases and substrates.
Xylanases are produced by a variety of organisms, including Aspergillus, Trichoderma, Streptomycetes, Phanerochaetes, Chytridiomycetes,
Ruminococus, Fibrobacter, Clostridium, Bacillus and Thermoascus,
using various industrial residues (Bastawde, 1992; Carvalho et al.,
2012a,b; Christov, Szakacs, & Balakrishnan, 1999; Gomes, Guez,
Martin, & Silva, 2007). The fungi produce xylanases, but they usually
co-secrete cellulases that can adversely affect the quality and purity
of the product (Balakrishnan, Dutta-Choudhary, Srinivasan, & Rele,
1992).
According to Abdel-Sater and El-Said (2001), the agro-industrial
wastes rice straw, wheat straw and sugarcane bagasse were used to
isolate 26 species using an agar medium containing xylan. Among
these species, the microorganisms Aspergillus avus, Aspergillus
niger, Penicillium chrysogenum, Penicillium corylophilum, Penicillium
funiculosum, Penicillium oxalicum and Trichoderma harzianum were
common among isolates from the three substrates. 93% of the isolates

81.0
52.0
77.0
15.9
13.8

12.9
60.0
37.1
86.6
82.4
68.5
36.8

5.8
2.7
3.7
3.1
2.8
2.7
1.8
1.5

6.7
5.7
0.65
0.79
0.88
4.0
1.7

Aachary and Prapulla (2009)

Aachary and Prapulla (2009)
Aachary and Prapulla (2009)
Sabiha-Hanim, Noor, and Rosma (2011)
Akpinar et al. (2009a)
Akpinar et al. (2009a)
Akpinar et al. (2009a)
Akpinar et al. (2009a)
Akpinar et al. (2009b)
Chapla, Pandit, and Shah (2012)
Brienzo, Carvalho, and Milagres (2010)
Samanta, Senani, et al. (2012)
Samanta, Senani, et al., (2012)
Samanta, Senani, et al., (2012)
Yang, Wang, Song, and Xu (2011)
Jayapal et al. (2013)

degraded xylan, and the genera Aspergillus, Fusarium, Penicillium and

Trichoderma showed the highest enzymatic activities on xylan. The
optimum conditions for the maximum yield of xylanases by T.
harzianum (50.6 U/ml) were 8 days of incubation at 35 C in submerged fermentation (SmF) using starch and maltose as the sole carbon substrates, NaNO3 and peptone as nitrogen sources and the pH of
the nutrient solution adjusted to 6.0.
The best substrates for extracting xylan to produce XOS and the
best xylanase microorganisms are not yet entirely clear, but there
are some residues and microorganisms that stand out. In Table 3,
some strains of fungi that produce xylanases using different substrates and their corresponding enzymatic activities are shown.
The combination of several fungal strains, substrates and different
bioprocess conditions show a wide range of xylanase production in
the literature. Submerged fermentations using wheat bran, corncobs,
oats and residues from a bleaching efuent were compared. The
higher levels of xylanase (547.4 U/ml) were reached when the medium with corncob was inoculated with Aspergillus foetidus ATCC 14916
(Christov et al., 1999).
Pretreatments were performed using acidic residues of cassava
hulls, corncobs, corn husks, oat hulls and sugar cane bagasse with
the liquid fraction used in the production of xylanases by Penicillium
janthinellum (Christov et al., 1999). The corncob (55.3 U/ml) and oat
hulls (54.8 U/ml) were the best inducers of xylanases, followed by
bagasse (23.0 U/ml) and corn straw (23.8 U/ml) (Oliveira et al.,
2006).
However, a study using the SmF of Penicillium janczewskii indicated that wheat bran was the best inducer (15.4 U/ml), followed by oat
bran (5.8 U/ml), corncob (5.3 U/ml), barley grain (4.9 U/ml) and
cane bagasse (3.1 U/ml) (Terrasan et al., 2010).

Table 3
Fungal producers of xylanases and enzymatic activities on different substrates and fermentation process.
Strains

Xylanase activity

Substrate

Fermentation

Reference

Cellulosimicrobium cellulans
Penicillium janczewskii
P. janczewskii
P. janczewskii
Penicillium janthinellum
P. janthinellum
Trichoderma reesei RUT-C30
Aspergillus awamori 2B.361 U2/1
A. awamori
A. awamori
Malbranchea ava MTCC 4889
Thermomyces lanuginosus ATCC 46882

0.7
15.4
5.8
3.1
55.3
23.8
10 U/ml
25 U/ml
4.4
100 U/g
15,000 U/g
5098 U/g

Cane bagasse
Wheat bran
Oat bran
Cane bagasse
Pretreated corncob
Pretreated cane bagasse
Pretreated cane bagasse
Pretreated cane bagasse
Grape marc
Wheat bran
Rice straw
Cellulose bagasse

SmF
SmF
SmF
SmF
SmF
SmF
SmF
SmF
SSF
SSF
SSF
SSF

Song and Wei (2010)

Terrasan et al. (2010)
Terrasan et al. (2010)
Terrasan et al. (2010)
Oliveira et al. (2006)
Oliveira et al. (2006)
Gottschalk et al. (2010)
Gottschalk et al. (2010)
Botella et al. (2005)
Botella et al. (2005)
Sharma, Chadha, and Saini (2010)
Christopher et al. (2005)

SmF: submerged fermentation; SSF: solid state fermentation.

U/ml
U/ml
U/ml
U/ml
U/ml
U/ml

U/g

A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

The grape bagasse was used in solid state fermentation (SSF) by

Aspergillus awamori (yield of 40.4 U/g in 24 h); when wheat bran
was used, the production was increased about 2.5 times in 72 h showing the importance of substrate in xylanase production (Botella et al.,
2005). The production of xylanase with other specie (Aspergillus
terreus MTCC 8661) in SSF showed a better performance (115 U/g)
in palm oil residue at 35 C after 60 h incubation (Lakshmi, Rao, Rao,
Hobbs, & Prakasham, 2009).
Some microorganisms really stand out in the production of
xylanases. Thermomyces lanuginosus ATCC 46882 cultivated in SSF at
45 C after 4 days of incubation in cellulose bagasse presented
5098 U/g of xylanase (Christopher et al., 2005). Sonia, Chadha, and
Saini (2005) working with the same specie and sorghum straw as
substrate showed 48,000 U/g substrate in SSF after optimization of
the parameters glycerol, inoculation level and ammonium sulfate
concentration. Moreover, another culture (Malbranchea ava MTCC
4889) had a high rate of xylanase (15,000 U/g) with rice straw in
SSF process, after 6 days of incubation at 45 C (Sharma et al., 2010).
The denition of the bioprocess is an important factor for xylanase
production. Some studies showed the highest level of xylanases using
the SSF process (Christopher et al., 2005; Sharma et al., 2010). Some
comparative studies of SSF and SmF bioprocess suggested 510
times more xylanases in the production by Penicillium brasilianum,
A. niger, and Melanocarpus albomyces when cultured under SSF
(Jorgensen, Morkeberg, Kristian, & Olsson, 2005; Narang, Sahai, &
Bisaria, 2001; Thygeson, Thomson, Schmidt, Jorgenson, & Olsson,
2003).
According to Sharma and Chadha (2011) aeration limitation and
the need for mechanical agitation in submerged processes, especially
batch process with stirred tank reactor are limiting factors in the
xylanase production by fungal aerobic process. Studies comparing different types of fermenters with A. niger demonstrated better xylanase
yield and productivity were obtained with bubble-column and an
air-lift bioreactor than in stirred-tank reactor.
One of the advantages of the SSF process is the low moisture content resulting in lower energy consumption and prevent bacterial contamination, besides better aeration compared with the submerged
cultivation (Leite et al., 2007; Pandey et al., 2000; Rodrigues, Dantas,
Pinto, & Goncalves, 2007). In the SSF process the best substrate is
one which generates spaces where the growth of hyphae may occur
in the presence of oxygen. In addition to this aspect, the fungus
shows good tolerance to low water activity and high osmotic pressure

81

conditions making this process more efcient for bioconversion of

solid substrates (Raimbault, 1998). Despite the difculties in the
scale up of SSF process when compared with SmF, it has been estimated that SSF is 100 times more economical for cellulase production as
compared to SmF (Antoine, Jacqueline, & Thonart, 2010).
The interaction between the organism and the source of substrate
may be crucial in qualitative and quantitative aspect of enzyme production, considering genetic aspects of biosynthesis and mechanisms
of enzyme induction. There are several types of xylanases and each
microorganism can show differences in enzymatic production (Hinz
et al., 2009) according to the structure of different substrates. Considering the fungi culture in various hemicellulose sources has been
shown differences in expression of functionally distinct xylanases
(Badhan, Chadha, & Saini, 2007).
5.2.1.2. Xylanases: general characteristics and applications. Hemicellulose, a heteropolymer with a complex structure mainly containing
xylan, requires the catalysis of various enzymes (Fig. 3) to complete
its hydrolysis (Biely, 1985).
Endo--1,4-xylanase (-1,4-D xylan xylanohydrolase, EC 3.2.1.8)
is essential for the depolymerization of xylan, acting on the main
chain and releasing oligosaccharides. -D-xylosidase (-1,4-xylan
xylohydrolase, EC 3.2.1.37) acts from the non-reducing end of
xylobiose or other XOS releasing xylose monomers. The removal of
side groups is catalyzed by -L-arabinofuranosidases (EC 3.2.1.55),
-D-glucuronidases (EC 3.2.1.139), acetyl xylan esterases (EC
3.1.1.72), ferulic acid esterases (EC 3.1.1.73) and p-coumaric esterases
(EC 3.1.1.73) (Belancic et al., 1995; Biely, 1985).
Xylanolytic enzymes can be classied as glycoside hydrolases belonging to the families 5, 7, 8, 10, 11 and 43; however, the most studied families are 10 and 11. These enzyme families differ in their
physicochemical properties, primary amino acid sequences, modes
of action and substrate specicities (Collins, Gerday, & Feller, 2005).
Studies have shown that the family 10 xylanases are not totally
specic for xylan and can be active on other substrates, such as oligosaccharides from cellulose. In addition, these enzymes are highly
specic for small xylo-oligosaccharides (Biely, Vransk, Tenkanen,
& Kluepfel, 1997). Members of this family have a molecular weight
greater than 30 kDa and a low isoeletric point.
In contrast, the enzymes of family 11 show low molecular weights
and high isoelectric points (Wong, Tan, & Saddler, 1988). This family
consists only of xylanases, and they are termed true xylanases. The

-4-O-Me-GluUA
Acetylxylan

Endo-1,4--xylosidase

-D-glucuronidase

-Xylose

-L-arabinofuranosidase
-araf.
Xylo-oligosaccharides

pcou./fer.

p-coumaric acid or
-Xylosidase

ferulic acid esterase

Fig. 3. Schematic structure of hemicellulose and the sites of xylanase reactions. The main chain consists of xylose residues with -1.4 linkages (Sunna & Antranikian, 1997).

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A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

enzymes of this group are only active on substrates containing D-xylose and long chains of xylo-oligosaccharides, they have low catalytic
versatility and the products of their action can be hydrolyzed by the
enzymes of family 10 (Biely et al., 1997). The family 10 xylanases produce XOSs that are smaller than those produced by xylanases from
family 11 (Maslen, Goubet, Adam, Dupree, & Stephens, 2007).
According to Song and Wei (2010), the demand for enzymes is
growing faster than ever, and this demand is a force driving research
on xylanases and cellulases. However, the production costs and low
yields are important problems for industrial applications. The incorporation of cheap sources (such as sugarcane bagasse, wheat straw
and corncob) in the culture media for the growth of microorganisms
and enzyme production should help to decrease the production costs
for the enzyme complexes. Consequently, several processes have
been and are being developed to use agro-industrial residues to generate products such as ethanol, proteins and enzymes using microorganisms (Lakshmi et al., 2009). Moreover, the bioconversion of these
substrates may help reduce the environmental impact caused by the
accumulation of waste (Camassola & Dillon, 2009).
Xylanolytic enzymes are highlighted due to their potential applications in the bioconversion of lignocellulose to sugars (Lakshmi et
al., 2009). They also have potential applications in industries in the
clarication of juices and vegetable oil extracts, as aids in the digestive acquisition of nutrients by pigs and birds that use cereal-based
diets, to improve our for bakery food products, as bleaching agents
in pulp and paper and in the saccharication of agricultural, industrial
and food wastes (Beg, Kapoor, Mahajan, & Hoondal, 2001; Moure
et al., 2006; Polizeli et al., 2005; Subramanian & Prema, 2002;
Techapun, Poosaran, Watanabe, & Sasaki, 2003). Furthermore,
xylanases can be used to produce high-value xylo-oligosaccharides
(Vzquez et al., 2000).
5.3. Enzymatic technology for XOS production
After the pretreatment of the LCM, the extracted xylan must be
treated by enzymatic hydrolysis (Ai et al., 2005; Akpinar et al.,
2009a; Brienzo et al., 2010; Carvalho et al., 2012a,b; Yang et al.,
2011) or an acid treatment (Akpinar et al., 2010; Rodrigues et al.,
2010). According to Sun et al. (2004), acid hydrolysis is not
recommended for this step, due to the production of undesirable
products such as furfural and hydroxymethylfurfural and the large
amount of monosaccharides released.
Currently, enzymatic hydrolysis is considered the best option to
produce XOS for the food industry (Aachary & Prapulla, 2009). Production of XOS is carried out by xylanases hydrolyzing -1,4 xylan
bonds. The enzymatic complex must have a low activity of exoxylanases (-xylosidases) to attenuate the production of xylose,
which inhibits XOS production (Vzquez et al., 2002). Moreover, to
increase XOS production, an endo-xylanase, which hydrolyses -1,4
bonds in the main chain of xylan, producing -anomers of XOS, and
enzymes that remove side groups must be used (Aachary &
Prapulla, 2009; Akpinar et al., 2010).
In the enzymatic process of oligosaccharide production, the enzyme can be added directly to the reaction medium (Akpinar et al.,
2010), immobilized (Jiang et al., 2004), produced in situ via microbial
fermentation or immobilized inside the biomass (Dorta, Cruz,
Oliva-Neto, & Moura, 2006; Oliva-Neto & Meno, 2009; Santos et al.,
2008). Fractionation of XOSs with different degrees of polymerization
can easily be accomplished by ultraltration to remove oligosaccharides with undesirable degrees of polymerization (Akpinar et al.,
2010).
Catalysis using immobilized and free endo-xylanases from Bacillus
halodurans for XOS production from corncob xylan was compared.
The conditions of the enzymatic reaction were as follows: 50 C,
pH 8.0, 12.8 U/g xylan and 2% of substrate in 24 h. The free enzyme
was more efcient in XOS production than the immobilized one.

The free enzyme converted xylan to higher-level oligomers with a degree of polymerization greater than 4, as well as 32.5% xylobiose and
xylotriose, suggesting that free xylanase worked on both longer (insoluble) and shorter (soluble) xylan chains. On the contrary, the
immobilized xylanase converted xylan to XOS with shorter lengths
and 25.2% of the mixture composed by xylobiose and xylotriose,
suggesting that the immobilized enzyme was more limited to acting
on shorter xylans (Lin, Tseng, & Lee, 2011).
In addition, other aspects inuence XOS yield and the composition
of oligosaccharides produced by enzymatic reactions, such as
xylanase type, xylan composition, the type of pretreatment, time
and other parameters of the reaction (Table 2).
Corncob has high potential to produce endo-xylanases and XOS.
Experimental studies using 233 U/g endo-xylanase from Aspergillus
oryzae MTCC 5154 and alkali-pretreated corncob (6%) at pH 5.4 and
50 C for 14 h, efciently exposed the xylan to the action of
endo-xylanases. Under these conditions, a high yield of XOS (79.7%),
corresponding 10.1 mg/ml, was obtained, and xylobiose (70.5%) was
the major component of the XOS mixture produced (Aachary &
Prapulla, 2009).
The pretreated xylan from triploid Populas tomentosa was converted
into XOS by a crude xylanase from Pichia stipitis. This enzyme converted
36.8% of the xylan to XOS, equivalent to 3.95 mg/ml, and 95% of the total
oligosaccharides were xylobiose, xylotriose and xylotetraose (Yang et
al., 2011).
XOS was produced by the catalysis of xylan extracted from corncob pretreated by mild alkali conditions (extraction yield of 17.9%)
using a partially puried xylanase from Aspergillus foetidus MTCC
4898. The enzymatic hydrolysis produced 60% (6.7 mg/ml) xylooligosaccharides, mainly xylobiose and xylotriose, after reaction of
8 h, with 20 U of xylanase/g xylan at 45 C (Chapla et al., 2012).
Moreover, the agroindustrial residue of palm oil was pretreated by
autohydrolysis and the solid residue obtained was hydrolyzed using
xylanases from Trichoderma viride. The sugar yield was: 13.9%
xylobiose, 1.97% xylotriose, 1.63% xylotetraose and 25.6% xylose
(Sabiha-Hanim et al., 2011).
The hydrolysis of different xylans was compared for XOS production using 200 U/g of xylanase from A. niger at 50 C over 24 h.
Under these conditions, the best production was with xylan from tobacco stalk (XOS yield of 13.8%), with a xylobiose yield of 7.95%
(1.59 mg/ml), a xylotriose yield of 5.9% (1.18 mg/ml) and yields of
other XOSs in lesser amounts (Akpinar et al., 2009a). The same
work compared the xylanases from different microorganisms. XOS
yield produced by xylanases from A. niger was greater than xylanases
from Trichoderma longibrachiatum on extracted xylan, with T.
longibrachiatum producing large amounts of monosaccharides.
The use of the endoxylanase from Trichoderma viridae (Sigma,
USA) for hydrolysis of xylan from grass extracted by alkaline hydrolysis produced approximately 2.8 mg/ml of XOS, xylobiose (1.7 mg/ml)
and xylotriose (1.1 mg/ml). The maximum yield of xylobiose (11%)
was obtained at pH 5.0 at 45 C, 87 U/g xylan in 10.1 h, and xylotriose
(7%) at pH 5.0 at 40 C and 66 U/g xylan in 16.5 h. (Samanta, Jayapal,
et al., 2012).
The maximum XOS production using xylan from sugar cane bagasse extracted by alkali and reacted with a commercial endoxylanase from T. viridae (Sigma, USA) was observed at 40 C, pH 4.0
with a dose of 13.3 U/g in 8 h. Under these conditions, 1.15 mg/ml
of xylobiose and 0.57 mg/ml of xylotriose were obtained. A response
surface analysis predicted the ideal conditions to be41 C, pH 4.9, enzyme dose 15.6 U/g and reaction time 19.2 h for maximizing
xylobiose yield, while for maximum xylotriose yield, the conditions
were 40 C, pH 4.13, dose 29.4 U/g and an incubation time of 18.1 h
(Jayapal et al., 2013).
However, the enzymatic hydrolysis with the xylan extracted from
sugar cane bagasse pretreated with alkali and peroxide showed a
maximum XOS conversion of 37.1%, after 96 h with 2.6% substrate,

A.F.A. Carvalho et al. / Food Research International 51 (2013) 7585

60 U/g of crude enzymatic extract from Thermoascus aurantiacus at

50 C and pH 5.0. The predicted maximum yield of xylobiose was
41.6%. The maximum concentration of XOS obtained in the experiments was 5.7 mg/ml (Brienzo et al., 2010). Xylan extracted from
cane bagasse with potassium hydroxide was hydrolyzed with
120 U/g of crude xylanase from Aspergillus fumigatus at 45 C and
pH 5.0. The enzymatic reaction yielded 40% of XOS (xylobiose and
xylotriose) after 48 h of reaction (Carvalho et al., 2012a,b). As exposed
in this review, there is sufcient space to develop XOS technology by
nding better substrates, xylanases, LCM pretreatments and xylan
treatments, aiming to improve the yield and quality of XOS and its
use as a functional ingredient in human and animal nutrition. From
the analysis of the cited research, it is possible to indicate the following: a) the pretreatment of xylan that is most often used and likely
the most efcient and safest is the alkaline hydrolysis, b) corncob is
the one of the best substrates for XOS production, due its high xylan
content, but other substrates can be used, such as sugar cane bagasse,
c) the best conditions for the xylanase reaction with xylan as the substrate are at least 8 h of reaction time, a temperature range of 40
50 C and pH 4.08.0, depending on the microorganism, and d) the
most frequent types of XOSs produced from LCM are xylobiose and
xylotriose, and mixtures of these XOS at 1.75.7 mg/ml with an XOS
yield of 8.537% can be produced from xylan using cane bagasse as a
substrate (Brienzo et al., 2010; Jayapal et al., 2013), and 2.810.2 mg/
ml and a yield of 3360% from corncob xylan (Aachary & Prapulla,
2009; Chapla et al., 2012; Lin et al., 2011). Other option is the rapid
thermo-acid hydrolysis of LCM, more simple but it requires a better
purication step to avoid the presence of toxins.
6. Conclusion
The production of XOS, which is a powerful and relatively new prebiotic, can contribute to the food and feed industries because it is an
important functional food ingredient with benets for human and animal health. In addition, the using lignocellulosic residues for XOS production represent an addition to the economic value of these products
and an increase in food production without the increase of agricultural
areas because XOS can be produced from agroindustrial residues. Research on XOS production demonstrates the need for improvements
in xylan extraction, including alkaline and acid hydrolysis pretreatments and XOS production by acid or enzymatic hydrolysis. For enzymatic hydrolysis, more studies of microorganisms and the production
and application of xylanases to improve XOS yields are necessary.
Studies of xylan sources are important to discover substrates richer
in this polymer, increase its extraction efciency and consequently
increase XOS production. Current research must focus on developing
viable and economic technologies from lignocellulosic residues reducing production cost and increasing the availability of XOS to develop a
wider market for XOS consumption. XOS production research is as
important as studies of applying XOS to replace antibiotics in feed
and to prevent important diseases in animals and humans.
Acknowledgments
The authors wish to thank Fundao de Amparo pesquisa do
Estado de So Paulo (FAPESP) and National Brazilian Research Foundation (CNPq) for nancial support.
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